Use of an aggressive, estrogen receptor -negative MCF-7 cell line variant, TMX2 -28, to study breast cancer: Expression of PLD1, MIG2, SKP2, and PALM in human breast carcinomas

• Main Author: Gozgit, Joseph M
• Language: ENG
• Published: ScholarWorks@UMass Amherst 2007
• Subjects: Molecular biology
• Online Access: https://scholarworks.umass.edu/dissertations/AAI3254930

We have used a novel estrogen receptor-(ER) negative, mixed basal/luminal, aggressive cell line, TMX2-28, as a model to study breast cancer. cDNA microarray comparison of TMX2-28 and its parent non-aggressive, ER-positive cell line, MCF-7, identified 1402 differentially expressed transcripts. Two-hundred upregulated transcripts were sorted by biological function and the expression of selected genes was assessed in TMX2-28 cells, MCF-7 cells, non-tumorigenic human mammary epithelial cells (HMECs), and thirty frozen human breast carcinoma specimens using real time RT-PCR. Four genes were selected for further studies: phospholipase D1 (PLD1), mitogen-inducible gene 2 (MIG2), S-phase kinase-associated protein 2 (SKP2), and paralemmin (PALM). PLD1 mRNA is expressed ten times more in TMX2-28 cells than in MCF-7 cells, and PLD1 is moderately expressed in non-tumorigenic HMEC lines 184, 184A1 and 184AA2. PLD1 mRNA levels were higher in breast tumors that expressed high mRNA levels of basal CKs 5 and/or 17. PLD1 protein was overexpressed in 10 of 42 (24%) breast tumors examined by IHC. Evaluating the expression of PLD1 with other signaling molecules, phospho-Akt and phospho-mTOR, we found that five PLD1-positive tumors were negative for phospho-Akt expression, but positive for phospho-mTOR expression. MIG2 is overexpressed 17-fold in TMX2-28 cells compared to non-aggressive MCF-7 cells and HMECs. MIG2 showed high mRNA levels in 30% of the human breast carcinoma specimens. siRNA-mediated suppression of MIG2 in TMX2-28 cells reduced TMX2-28 cell invasion by 48% compared to cells transfected with siRNAs against GAPDH. We also found that MIG2 protein was expressed in half of the breast tumors tested by IHC and showed heterogeneous expression among 21 tissues from reduction mammoplasty. SKP2 is overexpressed in TMX2-28 cells compared to MCF-7 cells by 13-fold, and SKP2 was not expressed in the HMEC lines. SKP2 mRNA levels were higher in breast tumors that expressed basal cytokeratins (CK) 5 and/or 17. Lastly, PALM is overexpressed in TMX2-28 breast cancer cells compared to MCF-7 and HMECs. We further evaluated the expression of PALM in human breast carcinomas and found that PALM is highly expressed on the plasma membrane of cells in roughly half of the breast tumors.